Project Summary Human papillomaviruses (HPV) are common sexually transmitted viruses and their infections are a significant health burden for the population. Persistent infection with high-risk HPV genotypes result in the development of a variety of human cancers, most prominently cervical, anal, and subtypes of head and neck cancers. HPV establishes persistent infection within the host through evasion of the immune system. One of the ways HPV accomplishes this is by manipulating Langerhans cells (LC), which are the antigen presenting cells at the site of infection that are responsible for initiating immune responses against epithelium invading viruses. The interaction of HPV with LC through the annexin A2 heterotetramer (A2t) and subsequent suppressive signaling events act as a braking mechanism that stops LC activation, resulting in incomplete LC maturation and prevention of an effective antiviral T cell immune response. Our long-term goals are to understand how HPV manipulates LC activation and antigen processing to evade the immune system and test new ways to treat HPV-associated diseases. AIM 1 will focus on the effects of utilizing a ?brakes off, gas pedal down? combination approach for activating LC exposed to HPV. This will be investigated through cell surface marker and cytokine analysis and functional immunological assays. AIM 2 will investigate the signaling pathways and A2t interaction partners associated with A2t-mediated suppression of LC immune function that are hijacked by HPV using biochemical and cell biological approaches. This will be analyzed through broad interrogation of phosphoprotein activation and cell signaling networks, antigen presenting cell specific profiling gene arrays, and a newly developed Single Molecule Pulldown (SiMPull) assay. AIM 3 will evaluate the contribution of A2t to LC Birbeck granule structure, function, and role in HPV antigen processing. This will be investigated through immuno-electron microscopic analysis of Birbeck granule components in LC, assessment of A2t protein knockdown effects on Birbeck granule formation, and elucidation of the effects of protein knockdown on the ability of LC to properly process and present HPV-derived peptides to HLA-matched T cell clones. Additionally, these aims will utilize innovative skin explant and 3-D culture systems to validate that the proposed HPV16-A2t interactions are important for LC biology in vivo. Collectively the proposed studies will provide deep insight into basic LC biology and function. In addition we will examine the effects of a novel two-pronged approach focused on preventing negative signaling events in LC while simultaneously providing a positive stimulus to activate LC as a novel strategy to treat HPV infections, thereby preventing HPV-associated cancers.